HRP960029A2 - Improved synthesis of cyclopropylacetylene - Google Patents

Description

State of the art

This case is related to Merck Case 187931A, which is a continuation in part of Merck Case 18793 filed Aug. 7, 1992, U.S.S.N. 07/926,607, and case 19344.

Retrovirus, designated as human immunodeficiency virus (HIV) is the etiological agent of a complex disease that includes progressive destruction of the immune system (acquired immunodeficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was formerly known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is reverse transcription of the RNA genome by virally encoded reverse transcriptase to produce DNA copies of HIV sequences, a step required in viral replication. Some compounds are known to be reverse transcriptase inhibitors and to be effective agents in the treatment of AIDS and related diseases, eg, azidothymidine or AZT.

Nucleotide sequencing of HIV shows the presence of the pol gene in one open reading frame /Ratner L et al., Nature, 313,277 (1985)/. Amino acid sequence homology provides evidence that pol sequences encode reverse transcriptase, endonuclease and HIV protease /Toh H et al., EMBO J., 4, 1267 (1985); Power MD et al., Science, 231, 1567 (1986); Pearl LH et al., Nature. 329. 351 (1987)/.

Applicants report a significantly improved synthesis of an HIV reverse transcriptase inhibitor of this structure

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named (-) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one hereinafter "Compound A". This compound is highly effective, even against HIV reverse transcriptase resistant to other AIDS antiviral compounds.

Applicants have developed a significantly improved synthesis of cyclopropylacetylene, an intermediate of Compound A. Previous methods used a two-step process with the use of corrosive reagents, with low overall yield. See, for example, Militzer HC et al., Synthesis, 998 (1993); Schoberth, W. et al., 703 (1972) /PCI5 and base/; Sherrod W et al, J.Am.Chem.Soc, 93:8, 1925-1940 (April 1971) f\2 on hydrazone/; Mikhailov and Bronovitskaya, Zh.Obshch. Khim., Vol. XXll, 195-201 (1952) /dibromide/. In contrast, this process is shorter than the previous ones, does not use corrosive reagents, and gives a total yield equal to or better than earlier processes. This procedure involves the cyclization of 5-halo-l-pentyne in a strong base.

Applicants have discovered that the successful outcome of this reaction requires obtaining an unstable dianion that is cyclized to cyclopropyl acetylene. Neither method of this type produces a dianion. On the contrary, the art indicates that various side reactions could occur, including replacement of chlorine by a base or deprotonated acetylene, or replacement of chlorine by a metal halide.

Brief description of the invention

An improved synthesis of cyclopropylacetylene, an intermediate of Compound A, is presented. This synthesis involves the cyclization of 5-halo-1-pentyne in a strong base. Compound A is useful in the inhibition of HIV reverse transcriptase (and its resistant strains), the prevention of HIV infection, the treatment of HIV infection and the treatment of AIDS and/or ARC, either as a compound, a pharmacologically acceptable salt (when appropriate) , an ingredient of a pharmacological preparation, whether or not in combination with other antiviral, anti-infective, immunomodulating agents, antibiotics or vaccines. Procedures for the treatment of AIDS, methods of prevention of HIV infection and methods of treatment of HIV infection are also presented.

Detailed description and preferred ways of realizing the invention

The process of this invention is aimed at obtaining cyclopropylacetylene, a reagent useful for adding a cyclopropylacetylene group to various antiviral agents and other ingredients of medical interest, especially HIV reverse transcriptase inhibitors. In the present invention, the process for obtaining cyclopropylacetylene comprises the following steps:

(a) mixing at least about 1.0 equivalents of a strong base in an aprotic solvent with a saturated equivalent of 5-halo-1-pentine in an aprotic solvent at a temperature between about -20°C and about 150°C;

(b) allowing the temperature of the reaction mixture to rise to a range between about 0 and 150°C and maintaining the temperature within this range for at least about 15 minutes, or until the cyclization is substantially complete;

(c) stopping the reaction with any source of protons.

In one way of realizing this invention, the process for obtaining cyclopropylacetylene comprises stages

(a) mixing at least about 1.0 equivalents of a strong base in an aprotic solvent with one equivalent of 5-halo-l-pentyne in an aprotic solvent at a temperature between approximately -20 and 150°C;

(b) allowing the temperature of the reaction mixture to rise to a range between about 0 and 150°C and maintaining the temperature within that range for at least about 15 minutes or until the cyclization is substantially complete;

(c) cooling the reaction mixture to a temperature between -30 and 50°C;

(d) stopping the reaction with any proton source.

In the next embodiment of this invention, the final stage of purification of the desired product, cyclopropylacetylene, is added.

The primary way of realizing this invention is a process for obtaining cyclopropylacetylene, which includes stages

(a) mixing between about 2.0 and 2.5 equivalents of n-butyllithium in cyclohexane with one equivalent of 5-chloro-1-pentyne in cyclohexane at about 0°C;

(b) heating the reaction mixture to about 75°C and maintaining the reaction at that temperature for at least 5 hours, or until the cyclization is substantially complete;

(c) cooling the reaction mixture to about 0°C; and

(d) quenching the reaction with saturated NH 4 Cl; and as an option

(e) purification of the desired cyclopropylacetylene product.

The process of this invention is a one-pot process, which begins by mixing one equivalent of S-halo-1-pentyne in an aprotic solvent with at least about 1.0 equivalents of a strong base in an aprotic solvent at a temperature between -20 and 150°C . A preferred range of equivalents of a strong base is between approximately 2.0 and 2.5 equivalents.

The primary starting material is 5-chloro-1-pentene. The preferred temperature for this mixing is in the range of about -20 to 25°C, preferably around 0°C. Before mixing, the aprotic solvent for the 5-halo-1-pentene may or may not be the same as the aprotic solvent for the strong base.

The strong base is selected from the group consisting of n-butyl lithium, sodium amide, sodium diethyl amide, sodium hydride, potassium hydride, sodium bis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, LDA, sec-butyl lithium , tert-butyl lithium and lithium tetramethyl piperidide. The primary strong base is n-butyl lithium.

Aprotic solvents selected from THF, 1,4-dioxane, MTBE, diethoxymethane, dimethoxyethane, cyclohexane, hexane and hexane with tetramethylene diamine. Primarily aprotic solvents cyclohexane.

Mixing a strong base with 5-halo-l-pentine is an exothermic reaction leading to cyclization. Cyclization occurs spontaneously. It is convenient to heat the reaction sufficiently to accelerate the cyclization. Primarily, the temperature for cyclization is in the range between approximately 50 and 80°C, preferably around 75°C. The higher the temperature, the shorter the time required to essentially complete the cyclization. It goes without saying that variations in temperature and incubation time can be readily determined by those skilled in the art.

When the cyclization is substantially complete, or at least sufficiently complete, the reaction mixture can optionally be cooled to a temperature between about -30 and 50°C, preferably to a temperature of about 0°C. A proton source is then added to stop the reaction. In this invention, the proton source is selected from saturated NH4Cl, HCI and H2SO4. The primary source of protons is NH4CI.

Finally, a purification step for the isolation of cyclopropylacetylene can be inserted at this point.

The reactions used to attach cyclopropylacetylene groups to the nuclei of other molecules involve well-known chemistry and are within the skill of the art. For the addition of cyclopropylacetylenic groups to aromatic substituents, palladium-catalyzed bonding is used. A displacement reaction is used to attach cyclopropylacetylenic groups to alkyl substituents.

The compounds of this invention have asymmetric centers and may occur, except when specifically indicated, as racemates, racemic mixtures, or as individual diastereomers, or enantiomers, all isomeric forms are included in this invention. The term (+/-) is intended to describe (+) optical isomers, (-) optical isomers or their mixtures.

When any variable (eg, aprotic solvent) occurs more than once in any degree, its definition in each occurrence is independent of the definition in every other occurrence. Also, combinations of substituents and/or variables are allowed only if such combinations give stable compounds.

As used herein, unless otherwise indicated, the term "alkyl" includes both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. "Halogen" or "halo" as used herein means fluoro, chloro, bromo and iodo.

In this invention, cyclopropylacetylene is obtained according to the following scheme.

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The total yield is above 65%. For comparison, the previous method using corrosive reagents gave about 42% yield and starts like this:

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as described in L.E. Hudson et al.. J. Am. Chem. Soc, 94, 1158 (1972) and W. Schoberth et al., Synthesis, 703 (1972).

Ingredient A can be synthesized by the following method

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Compound A is useful for obtaining and performing screening analyzes for antiviral compounds. For example, Compound A is useful for isolating enzyme mutants, which are excellent tools for searching for even more potent antiviral compounds. In addition, Compound A is useful in identifying or determining the binding site of other antiviral compounds for HIV reverse transcriptase, eg, by competitive inhibition.

Therefore Spoj A is a commercial product that is sold for these purposes.

Compound A is useful in inhibiting HIV reverse transcriptase, preventing or treating human immunodeficiency virus (HIV) infection, and treating consecutive pathological conditions such as AIDS. Treatment of AIDS or prevention or prevention or treatment of HIV infection is defined but not limited to the treatment of a wide range of conditions of HIV infection: AIDS, ARC (from Eng. AIDS related complex), symptomatic and asymptomatic and actual or potential exposure to HIV infection -om. For example, a compound of the present invention is useful for the treatment of HIV infection following suspected previous exposure to HIV through, e.g., blood transfusion, exchange of body fluids, bites, accidental needle sticks, or exposure to whole blood during a surgical procedure.

A particular advantage of Compound A is its strong inhibition of HIV reverse transcriptase resistant to other antiviral agents such as L-697,661, which is 3-(/)4,7-dichloro-1,3-benzoxazol-2-yl)-methyl/- amino-5-ethyl-6-methyl-pyridin-2(1H)-one; or L-696,229, which is 3-(2-(1,3-benzoxazol-2-yl)ethyl)-5-ethyl-6-methyl-pyridin-2(1H)-one; or AZT.

For these purposes, Compound A can be administered orally, parenterally (including subcutaneous injections, intravenous, intrasternal injection or infusion techniques), by inhalation nebulizer, or rectally in dosage unit preparations containing common non-toxic pharmacologically acceptable carriers, adjuvants and vehicles.

Thus, in accordance with the present invention, there is further provided a method of treatment and a pharmacological composition for the treatment of HIV infection and AIDS. Treatment involves administering to a patient in need of such therapy a pharmacological mixture consisting of a pharmacological carrier and a therapeutically effective amount of a compound of the present invention.

These pharmacological mixtures can be in the form of suspensions or tablets that are administered orally; nasal sprays, sterile injectable preparations such as aqueous or oily injectable suspensions or suppositories.

When administered in the form of oral suspensions, these compositions are prepared according to techniques well known in the art of making pharmacological preparations and may contain crystalline cellulose as a bulking agent, alginic acid or sodium alginate as a suspending agent, methyl cellulose as a viscosity-increasing agent, and for sweetening and flavoring known in the profession. As immediate release tablets of the active compound, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, fillers, disintegrators, diluents and lubricants known in the art.

When administered by nasal spray or inhalation, these compositions are prepared in accordance with techniques well known in the art of pharmaceutical preparation and may be prepared as solutions in physiological saline, using benzyl alcohol or other suitable preservatives, agents that promote absorption to increase biological availability, fluorocarbons or other means for dissolving or dispersing known to the art.

Injection solutions or suspensions can be prepared in accordance with the known manufacturing method, using suitable non-toxic ones. parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable wetting or suspending agents, such as sterile bland fixed oils, including synthetic mono and diglycerides and fatty acids including oleic acid .

When administered rectally in suppository form, these compositions can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters, or polyethylene glycols, which are solid at room temperature but liquid and/or soluble in water. the rectum releasing the drug. Compound A can be administered orally to humans in a dosage range of 1 to 100 mg/kg body weight in divided doses.

The first preferred dosage range is 0.1-10 mg/kg body weight orally in divided doses. Another preferred dose range is 0.1-20 mg/kg body weight in divided doses orally. For combination therapy with nucleoside analogues, the preferred dose range is 0.1-20 mg/kg body weight for compounds of the present invention administered orally in divided doses and 50 mg to 5 g/kg body weight for nucleoside analogues given orally in divided doses. It is understood, however, that the specific dose level and frequency of administration for each individual patient may differ and depend on various factors, including the activity of the particular applied compound, age, body weight, general condition, sex, diet, method and time of administration, excretion rate, the combination of drugs, the severity of the individual condition and the host being treated.

Example 1

Preparation of cyclopropylacetylene

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To a solution of 5-chloro-1-pentyne in cyclohexane (80 ml) at 0°C under nitrogen N2 was added n-butyllithium in cyclohexane (2.0 M, 122 ml). The mixture was heated to 75°C for 5 hours.

The addition of n-butyllithium to the alkyne is exothermic, the temperature being maintained below +5°C during these additions using an ice water bath.

The progress of the cyclization stage was monitored by HPLC. The reaction was considered complete when the analysis result was >90%. HPLC conditions: Phenyl column, CH3CN, water, phosphoric acid; 50:50:0.1 isocratic wash for 20 minutes. flow=1.0 ml/min, UV detection at 195 nm, starting material tR=7.5 min, cyclopropylacetylene tR=6.0 min. The product had a response factor 20 times higher than the starting material.

When the cyclization step was complete, the reaction was cooled to 0°C and quenched with saturated NH 4 Cl.

Analysis of the organic phase by HPLC showed 5.5 g of cyclopropylacetylene (85% yield).

The product was purified by fractional distillation through a 6" x 0.5" column packed with 4 mm diameter glass beads. The boiling point fraction between 45-75°C was collected.

This gave 4.2 g (65%) of cyclopropylacetylene as a colorless oil.

Example 2

Preparation of 4-chlorophenyl-pivalamide

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To a solution of 4-chloroaniline (76 g) in toluene (600 ml) was added saturated Na2CO3 (95 ml). The batch was cooled to 10°C and pivaloyl chloride (74 ml) was added dropwise over 45 minutes. The batch was stirred at 5-10°C for 60 minutes while the progress of the reaction was monitored by HPLC.

Addition of pivaloyl chloride to aniline was exothermic. HPLC conditions: C-8 column, CH3CN, water, phosphoric acid; washing gradient from 40:60:0.1 to 80:20:0.1 for 20 minutes, flow=1.0 ml/min. UV detection at 245 nm, starting materials tR=7.2 min, pivalamide tR=12.6 min.

The product was isolated by filtration and washed with D.I. water (3 x 75 ml) and dried in air under suction for 10 minutes. The product was dried in a vacuum dryer at 40°C by blowing N2 for 16 hours, whereby 108.5 g of product was obtained in the form of fine white needles (86%).

Example 3

Preparation of 4-chloro-keto-aniline

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In a 500 ml three-necked flask, pivalamide (10 g) was dissolved in dry THF (75 ml) and the mixture was cooled to 0°C. To this solution n-BuLi/hexane (2.5M, 38 ml) was added dropwise while the internal temperature was allowed to rise to +15°C. The batch was left to stand at 0°C for 2 hours.

Addition of the first equivalent of n-BuLi to pivalamide was highly exothermic. Exothermicity is controlled by the rate of addition.

Pure ethyl trifluoroacetate (6.7 ml) was added to the resulting light yellow suspension, while the internal temperature was allowed to rise to +10°C. The progress of the reaction is monitored by HPLC.

HPLC conditions: C-8 column, CH3CN, water, phosphoric acid; washing gradient from 40:60:0.1 to 80:20:0.1 during 20 minutes, flow=1.0 ml/min, UV detection at 245 nm, initial pivalamide tR=12.6 min, ketopivalamide tR=11 , 6 min. Typically 85A% product and 10-15A% unreacted pivalamide.

The reaction was quenched by addition of 6N HCl (10 ml) and D.I. of water (20 ml).

HPLC analysis showed 13.1 g (90%) of the product at this stage.

The solution is concentrated to approx. 50 ml in vacuo and washed with ethanol (50 ml) to remove hexane and THF. 6N HCl (40 ml) was added to the batch and the mixture was heated to reflux (80°C) for 1 hour.

HPLC analysis shows 85-95A% ketoaniline, 10A% unreacted pivalamide. Thus, the acylated material hydrolyzes while the unreacted pivalamide remains unchanged. The yield analysis at this stage was 7.78 g (74%).

The batch is concentrated to approx. 50 ml in vacuo during which time a precipitate (probably Hcl salt of the product) was formed. Distillation was stopped and the batch cooled to 0°C. After standing for 1 hour, the batch was filtered and washed with hexane (3 x 30 ml). Hexane washes unreacted pivalamide from the product. The solid is checked by HPLC to ensure that it is now completely removed. The filtrate and washings typically contain 1.2-1.5 g of product (8-12%). Most of the lost product was in the aqueous filtrate.

The salt was dried in a vacuum dryer at 40°C for 16 hours to give 10.4 g of a solid that was 71.4% pure by mass (70% yield). The salt is suspended in D.I. of water (260 ml) and neutralized to a pH of ca. 6-7 with 2N NaOH (15 ml).

It was critical not to bring the pH above 9 to degrade the product.

The resulting light yellow solid was isolated by filtration and washed with D.I. with water (2 x 25 ml). The product was dried in a vacuum dryer at 40°C for 16 hours to obtain 6 g of aniline with a mass purity of 96.6% (54% yield).

The product was further purified by recrystallization from hexane.

Example 4

Preparation of N-4-methoxybenzyl-keto-aniline

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Keto-aniline (15.5 g) activated 4Å molecular sieves (50 g) and toluene (75 ml) are charged into a 250 ml flask. The mixture was stirred at 23°C under N 2 for 24 hours. Analysis by HPLC showed a mixture of ca. 1:1 product and starting material.

HPLC conditions: C8 column, CH3CN, water, phosphoric acid; isocratic washing at 65:35:0.1 for 20 minutes, flow=1.0 ml/min, UV detection at 260 nm, toluene tp=5.7 min, initial keto-aniline tR=6.5 min, product tR = 15.0 min. Typically 25A% toluene is found. The reaction was batched with a fresh molecular sieve (40 g) and stirred for an additional 3 days at 23 °C. The reaction was considered complete when less than 2A% of the starting material remained.

The mixture was filtered through celite and washed with acetone (7 x 75 ml) until most of the yellow color was washed out of the celite. The filtrate was concentrated to give 27 g of a yellow-orange oil which solidified on standing. The solid was purified by dissolving in warm hexanes (100 ml). The batch was filtered and washed with cold hexanes (2x10 ml). The batch was air-dried by suction for 10 minutes, then dried in a vacuum dryer at 40 °C for 2 hours. This gave 20.5 g (86%) of light yellow powder.

Example 5

Obtaining amino alcohol

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Pyrrolidinyl ephedrine (264 mg) was dissolved in THF (2 ml) and the mixture was cooled to -5°C. Pure cyclopropylacetylene (0.11 ml) and sec-butyl lithium (2.0 ml) were added dropwise to the mixture at -5°C under N2. The mixture was allowed to stand at -5°C for 30 minutes and then cooled to -45°C.

Addition of sec-butyl lithium caused an exothermic reaction which was maintained between -5 °C to 0 °C at the rate of addition.

The ketone (175 mg) was dissolved in THF (1.0 mL) under N 2 and added to the anion mixture over 2-3 min while allowing the internal temperature to rise to -40 °C during the addition. The resulting light orange solution was left to stand for 60 minutes at - 40 °C and the reaction was stopped by the addition of 1M citric acid (3 ml) and ethyl acetate (3 ml). The reaction mixture was heated to room temperature and the layers were separated. The lower aqueous layer was extracted with ethyl acetate (3 ml). The combined organic layers were washed with 1 M citric acid (2x3 ml). The reaction mixture was analyzed by HPLC to determine the percentage of conversion and EE product.

HPLC conditions: C-8 column, CH3CN:water:phosphoric acid, isocratic wash 65:35:0.1 for 20 minutes, flow=1.0 ml/min, UV reading at 252 nm, starting material tR=12.8 min, product tR=10.3 min.

Chiral HPLC conditions: stationary phase amylose column, hexamisopropanol 85:15 isocratic elution, flow=1.0 ml/min, UV reading at 252 nm, starting material tR=4.9 min, major enantiomer tR=5.5 min, less represented enantiomer tR=25.0 min.

The enantiomeric excess was 98% and the reaction conversion 93% (6A% starting material). The yield of the analysis was 92%.

Example 6

Obtaining benzoxazinone

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The amino alcohol was dissolved in THF (15 mL) and cooled to -10°C under N 2 . Triethylamine (5.4 ml) and phosgene in toluene (4.6 ml) were added to the mixture. The addition of phosgene caused an exothermic reaction, which was maintained below 20°C at the rate of addition. The progress of the reaction was monitored by HPLC and the reaction was typically completed within 15 minutes.

HPLC conditions: C-8 column, CH3CN:water:phosphoric acid, elution gradient from 50:50:0.1 to 90:10:0.1 for 20 minutes, flow=1.5 ml/min, UV reading at 252 nm , starting material tR=14.6 min, product tR= 16 min.

The reaction was cooled to 0°C and quenched with ice-cold water (15 ml) and ethyl acetate (20 ml). A saturated salt solution was used to break the emulsion. The organic layer was removed and the aqueous layer was extracted with ethyl acetate (15 ml). The combined organic solutions were washed with 1M citric acid (40 ml) and brine (25 ml).

The organic solution was dried (Na 2 SO 4 ) and concentrated in vacuo to give 3.8 g of a brown oil.

This product was crystallized from 5:1 hexane:ethyl acetate (25 ml), cooled to 0°C, allowed to stand for 1 hour and filtered.

The cake was washed with cold 5:1 hexane:ethyl acetate (2x5 ml). The cake was air dried with suction to give 2.9 g (85%) of a light orange solid.

Example 7

Obtaining compound A

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The p-methoxybenzyl protected compound A was dissolved in CH3CN (15 ml). To this solution was added a solution of cerium ammonium nitrate (4.4 g) in water (5 ml). Reactions were typically complete after 2 hours at 23 °C as determined by HPLC.

HPLC conditions: C-8 column, CH3CN:water:phosphoric acid, elution gradient from 50:50:0.1 to 90:10:0.1 over 20 minutes, flow=1.5 ml/min, UV reading at 252 nm, starting material tR==16.0 min, product tR=9.0 min.

The reaction mixture was diluted with D.I. water (5 ml) and concentrated to approx. 1/2 volume. The product was isolated from the obtained aqueous layer with ethyl acetate (2 x 15 ml). The combined organic extracts were washed with D.I. water (2 x 10 ml) and saturated salt solution (10 ml). The organic extracts were concentrated in vacuo to give a yellow gum.

The product was isolated by chromatography on silica gel.

Example 8

N-(4-chlorophenyl)-2,2-dimethylpropanamide

4-Chloroaniline (127.57 g, 1 mol), 1200 mL of CHCl 3 , and 1200 mL of saturated aqueous Na 2 CO 3 were added to a 5 L three-necked flask with a vertical stirrer. An addition funnel was attached to the flask and charged with 2,2-dimethylpropanoyl chloride (129 mL, 1.05 mol). Acid chloride was added dropwise to the vigorously stirred mixture over 1 hour. The resulting mixture was stirred at room temperature for another 23 hours. Some product separated from the mixture in the form of white crystals. These crystals were collected by filtration. The filtrate was transferred to a separatory funnel and the layers were separated. The chloroform layer was washed with water and brine. Drying (MgSO4), filtration and removal of solvent in vacuo afforded additional product. The two product portions were combined and recrystallized from hot EtOAc-hexane to give 185.6 g of N-(4-chlorophenyl)-2,2-dimethylpropanamide as a white crystalline solid.

Example 9

(-)6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one

(Compound A) and (+) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-1,3-benzoxazin-2-one

Grade A 2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol

A solution of bromomagnesium cyclopropylacetylide was obtained from 23 g of cyclopropylacetylene (0.384 mol) in 250 ml of THF, by the dropwise addition of 116 ml of a 3.OM solution of ethylmagnesium bromide in ether (0.384 mol) over 1 hour. This solution was kept at 0°C for one hour and then at 40°C for 3 hours. To this solution, cooled again to 0°C, 1-(2-amino-5-chlorophenyl)-2,2,2-trifluoromethylethanone (0.0696 mol) was added as a solid in portions over 5 minutes. The reaction was allowed to stir at 0°C for 1.5 hours. The reaction was stopped at 0°C by dropwise addition of 700 ml of a saturated aqueous solution of ammonium chloride. The mixture was extracted with 2 x 400 ml portions of ethyl acetate, the combined organic phases were washed with brine and dried over MgSO4. Removal of the drying agent and solvent left a yellow solid. This material was recrystallized from boiling hexanes (100 ml final volume) to give 14.67 g of 2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2- ol. The second part of the yield (2.1 g) was obtained by concentrating the mother liquors. mp: 153-154 °C. 1H-NMR (CDCl3): δ 0.84 (m, 2H), 0.90 (m. 2H), 1.38 (m, 1H), 4.50 (br s, 3H), 6.69 (d ,J=8.5 Hz, 1H), 7.13 (dd,J= 2.5, 8.5 Hz, 1H), 7.55 (d,J=2.5 Hz, 1H).

Stage B: (+)6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1 -

benzoxazin-2-one

A solution of 2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1-trifluoro-3-butyn-2-ol (15.00 g, 0.0518 mol) and 41.98 g (0.259 mol) of 1,1'-carbonyldiimidazole, in 250 ml of dry THF, was stirred under argon at 55 °C for 24 hours. The solvents were removed on a rotary evaporator and the residue was partitioned between 500 ml of ethyl acetate and 400 ml of water. The layers were then separated and the aqueous phase was re-extracted with ethyl acetate. The combined ethyl acetate extracts were washed with 2 x 200 ml of 2% aqueous HCl, saturated NaHCO3 solution, and saturated saline. By drying over MgSO4, filtration and removal of the solvent in vacuo, 12.97 g of analytically pure (+) 6-chloro-4-cyclo-propylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazine-2 were obtained. - in the form of white crystals. mp: 178-180 °C. 1H-NMR (CDC^):

0.85(m, 2H). 0.94 (m, 1H), 1.40 (m, 1H), 6.81 (d,J=8.5 Hz, 1H), 7.37 (dd,J=2.5, 8.5 Hz, 1H), 7.49 (dJ=2.5 Hz, 1H), 8.87 (br s, 1H).

Grade C 6-chloro-1-(1S)-camphanoyl-4-cyclopropylethynyl-4-trifluoromethyl-1,4-

dihydro-2H-3.1-benzoxazin-2-one

A solution containing (+)6-chloro-4-cyclopropyl-ethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one (12.97 g, 0.041 mol), 4-dimethylaminopyridine (1.02 g, 0.0083 mol), and (-) camphanic acid chloride (14.22 g, 0.06556 mol) in 350 ml of dry dichloromethane stirred under argon in an ice bath, triethylamine (22, 84 ml, 0.164 mol). The cooling bath was removed and the reaction was allowed to proceed at room temperature. After 75 min, the reaction was judged complete by thin layer chromatography (SiO2, 4% EtOAc in CHCl3) and the solution was diluted with 500 ml CHCl3 and then washed with 10% citric acid (2x), water (1x) and brine (1x) ). Drying (MgSO4), filtration and removal of the solvent in vacuo left a colorless foam. This material was triturated with 200 mL of boiling hexane. Upon cooling to room temperature, the desired diastereoisomeric camphanate imide precipitated. The solid was collected on a frit, washed with a little cold hexanes and dried in vacuo to give 7.79 g of 6-chloro-1-(1S)-camphanoyl-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro- 2H-3,1-benzoxazin-2-one in the form of white crystals. mp: 164-165°C. HPLC purity: 99.2% @ 254 nm. 1H-NMR(CDCl3): δ 0.77 (s, 3H). 0.86-0.96(m, 4H) 1.08(s, 3H), 1.19(s, 3H). 1.44(m, 1H), 1.95(m, 1H), 2.51 (m, 2H), 7.42 (dd,J=2.4, 9.0 Hz, 1H), 7.63 (m , 2H).

Grade D: (-) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1 -

benzoxazin-2-one (ingredient A)

6-Chloro-1-(1S)-camphanoyl-4-cyclopropylethynyl-4-trifluoromethyl,2-dihydro-4(H)-3,1-benzoxazin-2-one (7.50 g, 0.01512 mol) is dissolved in 150 ml of n-butanol at 60°C in an argon atmosphere. 10 ml of 1N Hcl was added to this solution. This solution was left at 60 °C for 72 hours. The mixture was neutralized with aqueous NaHCO 3 and the n-butanol was removed in vacuo. The residue was dissolved in 150 ml of THF and treated with 50 ml of 2N LiOH for 3 h at room temperature. This mixture was diluted with ethyl acetate and washed with two portions of water and one portion of brine. Drying (MgSO4), filtration and removal of the solvent in vacuo afforded a white solid. This material was recrystallized from hot hexane to give 3.43 g of (-) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one as white crystals. . mp: 131-132°C; [α]D20=84.7°C (CHCl3, c=0.005 g/ml); 1H-NMR (CDCl 3 ): δ 0.85 (m, 2H). 0.94 (m, 2H), 1.40 (m, 1H); 6.81 (d, J=8.5 Hz, 1H), 7.37 (dd, J= 2.5, 8.5 Hz, 1H), 7.49 (d, J= 2.5 Hz, 1H), 8.87 (number s, 1H).

Grade E (+) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-

benzoxazin-2-one

The mother liquors from step C were purified by silica gel column chromatography using 10% ethyl acetate in hexanes as eluent. The pure undesired diastereoisomer (colorless foam) is hydrolyzed according to step D. Enantiomeric benzoxazinone. (+) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one obtained as white crystals. mp: 131-132 °C; [α]D20=+84.4° (CHCl3, c=0.005 g/ml); 1 H-NMR (CDCl 3 ): δ 0.85 (m, 2 H), 1.40 (m, 1 H), 6.81 (d, J =8.5 Hz, 1 H). 7.37 (dd,J= 2.5. 8.5 Hz, 1H), 7.49 (d, =2.5 Hz, 1H), 8.87 (brs. 1H)

Reverse transcriptase analysis

The assay measures the incorporation of tritiated deoxyguanosine monophosphate by recombinant HIVa reverse transcriptase (HIV RTR) (or other RT). into acid-precipitable cDNA on the Km values of dGTP and poly r(C)-oligo d(G)12-18. The inhibitors of this invention inhibit this incorporation.

Analyzes were performed in 55 mM Tris (pH 8.2)-30 mM Kcl-30 mM MgCl2-1 mM dithiothreitol-20 μq or rC:dG12-18 (Pharmatia) at ml-(mM [3H]d GTP (New England Nuclear-0.01% Triton X-100-50 mM ethylene glycol-bis(β-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-1 mg bovine serum albumin per ml. After 60 minutes of incubation at 37°C, acid-precipitable material was collected on glass fiber filters using a semi-automatic cell harvester. Bacterial cell extracts containing RT were diluted within the linear range of the assay, and their activity was measured in the presence of inhibitors. and without them. Purified HIV-1 RT heterodimer produced on E. coli also served as a control. Results were determined as the inhibitor concentration giving 50% inhibition (IC50 wt), in nanomoles/liter. Compound A gave an IC50 wt of 2 nm.

For the analysis of the double mutant (dm), A17 RT was used for analysis. A 17 RT is resistant to various aminopyridones, as described in Nunberg, J.H. et al., J. Virol., 65, 4887 (1991). Results were measured as IC50 in nanomoles per liter. Compound A gave an IC50 of 85 nM.

Analysis of cell proliferation

Inhibition of HIV spread and cell culture measured according to Nunberg, J.H. et al.. J.Virol., 65, 4887 (1991), In this assay, MT-4 T-lymphoid cells were infected with HIV-1 virus (wild type unless otherwise stated) using a predetermined inoculum and cultures were incubated for 24 hours. At this time, ≤1% of cells were positive by indirect immunofluorescence. Cells were then washed extensively and distributed into 96-well culture plates. Serial 2-fold dilutions of inhibitor were then added to the wells and cultures continued for 3 subsequent days. On the fourth day after infection, 100% of the cells in the control cultures were infected. Accumulation of HIV-1 p24 directly correlated with viral spread. The inhibitory concentration for cell culture is defined as the inhibitory concentration in nanomoles/liter that reduced the spread of infection by at least 95% or CIC95.

While the foregoing specification teaches the principles of the present invention by way of illustrative examples, it is understood that the practice of the present invention includes all customary variations, adaptations or modifications, as they appear within the scope of the following claims and their equivalents.

Claims (11)

1. Process for obtaining cyclopropylacetylene, characterized by the fact that it includes stages a) mixing at least about 1.0 equivalents of a strong base in an aprotic solvent with one equivalent of 5-halo-1-pentine in an aprotic solvent at a temperature between approximately -20 and approximately 150°C; b) allowing the temperature of the reaction mixture to rise to a range between about 0 and about 150°C and maintaining the temperature within this range for at least about 15 minutes, or until the cyclization is substantially complete; and c) stopping the reaction with any source of protons. 2. The process for obtaining cyclopropylacetylene, characterized by the fact that it includes stages a) mixing at least about 1 equivalent of a strong base in an aprotic solvent with an equivalent of 5-halo-1-pentine in an aprotic solvent at a temperature between approximately -20 and 150°C; b) allowing the temperature of the reaction mixture to rise to a range between about 0 and about 150°C and maintaining the temperature within this subrange for at least about 15 minutes, or until the cyclization is substantially complete; c) cooling the reaction mixture to a temperature between approximately -30 and approximately 50°C; d) stopping the reaction with any source of protons. 3. The process according to any one of claims 1 or 2, characterized in that the final stage is added, which is the purification of the desired product, cyclopropylacetylene. 4. The method according to any one of claims 1-3, characterized in that the strong base is selected from the group consisting of n-butyl lithium, sodium amide, sodium diethyl amide, sodium hydride, potassium hydride, sodium bis(trimethylsilyl)amide , potassium bis(trimethylsilyl)amide, LDA, sec-butyl lithium, tert-butyl lithium and lithium tetramethyl piperidide. 5. The method according to claim 4, characterized in that the strong base is n-butyl lithium. 6. Process according to any one of claims 1-3, characterized in that the aprotic solvent is selected from THF, 2,5-dimethyl THF, 1,4-dioxane, MTBE, diethoxymethane, dimethoxyethane, cyclohexane, hexane and hexane with tetramethylene diamine . 7. The method according to claim 6, characterized in that the aprotic solvent is cyclohexane. 8. The method according to any one of claims 1-3, characterized in that the proton source is selected from saturated NH4CI, HCl and H2SO4. 9. The method according to any one of claims 1-3, characterized in that 5-halogen-1-pentyne, 5-chloro-1-pentyne. 10. Process for obtaining cyclopropylacetylene, characterized in that it includes stages a) mixing between about 2.0 and about 2.5 equivalents of n-butyllithium in cyclohexane with one equivalent of 5-chloro-1-pentyne in cyclohexane at about 0°C; b) heating the reaction to about 75 °C and maintaining the reaction at that temperature for about 5 hours, or until the cyclization is essentially complete; c) cooling the reaction mixture to approximately 0°C; d) stopping the reaction with saturated NH4CI. 11. The method according to claim 10, characterized in that the final step is added, which is the purification of the desired cyclopropylacetylene product.